Currently, most agents under active clinical investigation for the treatment of the human immunodeficiency virus (HIV), are virustatic compounds that target the viral reverse transcriptase (RT). The primary objectives of this proposal are (1) to study in detail the mechanisms of viral drug resistance and (2) to identify new strategies for overcoming drug resistance. The most direct and clinically relevant means of pursuing the aforementioned aims is the planned longitudinal virus isolation from selected patients on antiviral therapy. The isolates will be screened serially in terms of their sensitivity to the drug employed as well as to other antiviral compounds. Given that most of the anti-HIV compounds target the RT, it is crucial to know whether the mechanism of clinical resistance is the result of an alteration in the RT with respect to these compounds, their active metabolites or an alteration in the host's metabolism. To characterize further the molecular basis of the enzyme resistance, the viral RNA will be amplified via the PCR technique and further cloned and packaged via a plasmid pkk 322-2 vector to transform E. coli JM 109 cells. The extracted RT will be examined with regard to its kinetic interaction with antiviral compounds or their metabolites. DNA sequencing of the resistant recombinants' RT will be performed to assess whether point mutations or a 'hot-spot' on the viral genome renders resistance or cross-resistance to compounds exerting their action through RT inhibition. To study the possible host factors involved in the development of resistance, the metabolism of the nucleoside analogues as harvested from both patient peripheral blood mononuclear cells (PBMC) and plasma will be examined. The plasma levels of naturally occurring nucleosides which can overcome the therapeutic action of nucleoside analogues will also be explored. In summary, these studies should provide an understanding of the molecular mechanisms responsible for the emergence of clinically resistant HIV to any given drug, and provide a clinically relevant rational for selecting and developing alternative agents to overcome that resistance.